Stimulation Of Human Mast Cells Research Articles

Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells

DJ-1 is a redox-sensitive protein with multiple roles in cell homeostasis, levels of which are altered in patients with mast cell (MC)-related disorders. However, whether DJ-1 can regulate human MC function is unknown. We sought to investigate the potential role of DJ-1 in the responses of human MCs to antigen stimulation. DJ-1 was silenced in human CD34+-derived MCs and in the LAD2 MC line by using lentiviral short hairpin RNA constructs. Release of β-hexosaminidase, prostaglandin D2, and GM-CSF and changes in reactive oxygen species levels were measured after FcεRI engagement. Enzymatic assays, sucrose density gradient centrifugation, immunoprecipitation, dot and Western blotting, and confocal imaging were performed for signaling, cellular localization, and coassociation studies. DJ-1 knockdown substantially reduced mediator release, as well as Lyn kinase and spleen tyrosine kinase activation and signaling through mechanisms that appeared largely unrelated to DJ-1 antioxidant activity. Following FcεRI activation, nonoxidized rather than oxidized DJ-1 translocated to lipid rafts, where it associated with Lyn, an interaction that appeared critical for maximal Lyn activation and initiation of signaling. Using purified recombinant proteins, we demonstrated that DJ-1 directly bound to Lyn but not to other Src kinases, and this interaction was specific for human but not mouse proteins. In addition, DJ-1 reduced Src homology 2 domain-containing phosphatase 2 phosphatase activity by scavenging reactive oxygen species, thus preventing spleen tyrosine kinase dephosphorylation and perpetuating MC signaling. We demonstrate a novel role for DJ-1 in the early activation of Lyn by FcεRI, which is essential for human MC responses and provides the basis for an alternative target in allergic disease therapy.

Virus stimulation of human mast cells results in the recruitment of CD56+T cells by a mechanism dependent on CCR5 ligands

The trafficking of effector cells to sites of infection is crucial for antiviral responses. However, the mechanisms of recruitment of the interferon-γ-producing and cytotoxic CD56(+) T cells are poorly understood. Human mast cells are sentinel cells found in the skin and airway and produce selected proinflammatory mediators in response to multiple pathogen-associated signals. The role of human mast cell-derived chemokines in T-cell recruitment to virus infection was examined. Supernatants from primary human cord blood-derived mast cells (CBMCs) infected with mammalian reovirus were examined for chemokine production and utilized in chemotaxis assays. Virus-infected CBMCs produced several chemokines, including CCL3, CCL4, and CCL5. Supernatants from reovirus-infected CBMCs selectively induced the chemotaxis of CD8(+) T cells (10±1%) and CD3(+)CD56(+) T cells (19±5%). CD56(+) T-cell migration was inhibited by pertussis toxin (65±9%) and met-RANTES (56±7%), a CCR1/CCR5 antagonist. CD56(+) T cells expressed CCR5, but little CCR1. The depletion of CCL3, CCL4, and CCL5 from reovirus-infected CBMC supernatants significantly (41±10%) inhibited CD56(+) T-cell chemotaxis. This study demonstrates a novel role for mast cells and CCR5 in CD56(+) T-cell trafficking and suggests that human mast cells enhance immunity to viruses through the selective recruitment of cytotoxic effector cells to virus infection sites. These findings could be exploited to enhance local T-cell responses in chronic viral infection and malignancies at mast cell-rich sites.

Characterization of ERK Activation in Human Mast Cells Stimulated by Contact with T Cells

Close physical proximity between mast cells and T cells has been demonstrated in several human conditions. We have identified and characterized a novel mast cell activation pathway initiated by contact with T cells, and showed that this pathway is associated with cytokine release. It has been shown recently that Ras is activated in this pathway. Thus, in the present study we further explore the downstream events associated with Ras activation and cytokine release in human mast cells stimulated by contact with T cells. ERK activation in human mast cells stimulated by either contact with T cells or by crosslinking the FC epsilon receptor was studied. Photobleaching experiments were used to study ERK localization. Enzyme linked immunosorbent assay was used to study the cytokine release by human mast cells. We show that stimulation of human mast cells by contact with activated T cells results is sustained ERK activation. Furthermore, sustained ERK activation in these cells is associated with increased dwell time at the nucleus and with IL-8 release. Interestingly, when mast cells were stimulated by crosslinking the FC epsilon receptor I, ERK activation was transient. ERK activation was associated with a shorter dwell time at the nucleus and with TNF-alpha release. Thus, retaining ERK in the nucleus might be a mechanism utilized by human mast cells to generate different cytokines from a single signaling cascade.

Stimulation of Human Mast Cells by Heterotypic Adhesion to T Cells Leads to Activation of Ras via. RasGRP1

Stimulation of Human Mast Cells by Heterotypic Adhesion to T Cells Leads to Activation of Ras via. RasGRP1

Genome activity profiling of monomeric-IgE and Fc-epsilon-RI-aggregation on human mast cells reveals a complex network of genes involved in inflammatory responses.

AbstractMast cell activation, mediates type-1 allergic responses, one of the most powerful reactions of the immune system. However, mast cells activation is becoming increasingly linked to inflammatory, autoimmunity, and to adaptive immunity by regulating T-cell activation.Here we analyzed the gene expression pattern in IgE-sensitized and FcεRI aggregation on human mast cells. Our data revealed coordinated changes in gene expression. We observed increased expression of gene-transcripts involved in allergic, innate and adaptiveimmune responses. Among the most prominent findings is the increased expression of transcripts encoding for MIP3a, SPARCL1, AREG, IL18, CCL1, TNFRSF9, IL1b, CX3CR1, PTGER3, MIF, MMP12, ADORA3, IL8RB, and other genes involved in innate and cellmediatedimmunity. These results represent a substantial advance in our understanding of the genome-wide effects triggered by “passive sensitization” or active stimulation of human mast cells, and how this relate to mast cells involvement not only in allergicresponses but also in innate and adaptive immunity.

Genome-wide gene expression profiling of human mast cells stimulated by IgE or FcεRI-aggregation reveals a complex network of genes involved in inflammatory responses

BackgroundMast cells are well established effectors of IgE-triggered allergic reactions and immune responses to parasitic infections. Recent studies indicate that mast cells may play roles in adaptive and innate immunity, suggesting an innovative view of the regulation of immune responses. Here, we profiled the transcriptome of human mast cells sensitized with IgE alone, or stimulated by FcεRI aggregation.ResultsOur data show that among 8,793 genes examined, 559 genes are differentially regulated in stimulated mast cells when compared with resting/unstimulated mast cells. The major functional categories of upregulated genes include cytokines, chemokines, and other genes involved in innate and adaptive immune-responses. We observed the increased expression of over 63 gene-transcripts following IgE-sensitization alone. Our data was validated using Real-Time-PCR; ELISA and western blot. We confirmed that IgE alone does not trigger mast cell-immediate responses, such as calcium signals, degranulation or protein-phosphorylation.ConclusionThis report represents a substantial advance in our understanding of the genome wide effects triggered by "passive sensitization" or active stimulation of human mast cells, supporting mast cells' potential involvement in a wide range of inflammatory responses.

Human T lymphocytes and mast cells differentially express and regulate extra- and intracellular CXCR1 and CXCR2.

CXCL8 plays a major role in cell recruitment to sites of inflammation. Apart from neutrophils, little is known, however, about the cellular distribution and regulation of CXCL8 receptors in cells involved in acquired and adaptive immune responses. In previous studies, we have demonstrated the extracellular expression and function of CXCR1/2 on mast cells and also detected an intracellular pool of CXCR1/2. Here, we have addressed the question of receptor regulation during stimulation of human mast cells (HMC-1 cell line) and have studied T cells in comparison. Cell permeabilization was performed to detect both surface and possible intracellular receptor pools. HMC-1 cells stained positive for both receptors on the cell surface (CXCR1, 50%; CXCR2, 51%) and also after cell permeabilization (CXCR1, 86%; CXCR2, 74%). Similarly, T cells exhibited both cell-surface receptor expression (CXCR1, 30%; CXCR2, 23%) and higher total receptor expression (CXCR1, 50%; CXCR2, 36%), although overall values were lower than that in HMC-1 cells. On immunoblot, molecular weights of extra- and intracellular receptors on mast cells were the same, excluding altered receptor glycosylation. On stimulation with phorbol 12-myristate 13-acetate plus calcium ionophore, a time-dependent decrease of surface-membrane receptors was observed in both cell types, while total receptor remained the same, suggesting that receptor shedding is not involved. The kinetics of membrane receptor internalization and replenishment differed for the two cell types. Furthermore, receptor internalization was associated with decreased F-actin polymerization, a basic prerequisite for cell migration. These findings demonstrate for the first time the expression of extra- and intracellular CXCR1/2 receptors on T cells and delineate the dynamics of CXCR1/2 receptors on mast cells and T cells. Furthermore, they suggest a cell-type-specific and finely tuned regulation of chemokine responses at the receptor level in the context of inflammation.

FcϵRI and TLR2-dependent cytokine generation by human mast cells utilize both convergent and divergent signaling pathways

Mast cells (MCs), effector cells of allergic reactions through FcϵRI, also initiate innate immune responses through pattern recognition receptors such as Toll-like receptors (TLRs). Stimulation of human MCs (hMCs) through either FcϵRI or TLR2 initiates cytokine gene expression; whether this process involves separate or distinct downstream signaling in hMCs is not known. Cord blood derived hMCs generated IL-5, IL-6, IL-13, TNF-α, MIP-1β, I-309, Rantes and MCP1 in response to both FcϵRI cross-link with α-IgE (1 μg/ml) and TLR2-dependent activation with staph peptidoglycan (PGN, 10 μg/ml). IL-4 priming substantially enhanced cytokine generation (MIP-1β, IL-5 and TNF-α) to both activating ligands. TNF-α generation through FcϵRI was markedly sensitive to the calcineurin inhibitor cyclosporine (CsA), (5 μg/ml) (93 ± 2 %inhibition) and UO126 (5 μg/ml), an inhibitor of MEK/ERK signaling (88 ± 5% inhibition) whereas PGN-dependent TNF-α production was less sensitive to these inhibitors (20 ± 15% and 50 ± 11% respectively). Both FcϵRI and TLR2-dependent stimulation elicited NF-κB-dependent transcription, as evidenced by blockade of TNF-α secretion by the IKK2 inhibitor PS1145 (72 ± 18 and 59 ± 11% inhibition respectively), which also attenuated nuclear translocation of p65 in the IgE and PGN samples. P38 inhibitor SB203580 (10 μg/ml) also inhibited cytokine generation through both the receptors. We conclude that hMCs uses both convergent and divergent pathways to generate cytokines in response to stimuli pertinent to allergy and to innate host protection, respectively. Furthermore, IL-4 amplifies cytokine generation through both receptor systems, suggesting an effect at the level of a common post-receptor signaling event.

Dichotomy of Ca2+ Signals Triggered by Different Phospholipid Pathways in Antigen Stimulation of Human Mast Cells

Mast cell activation triggers Ca(2+) signals and the release of enzyme-containing granules, events that play a major role in allergic/hypersensitivity reactions. However, the precise molecular mechanisms that regulate antigen-triggered degranulation and Ca(2+) fluxes in human mast cells are still poorly understood. Here we show, for the first time, that a receptor can trigger Ca(2+) via two separate molecular mechanisms. Using an antisense approach, we show that IgE-antigen stimulation of human bone marrow-derived mast cells triggers a sphingosine kinase (SPHK) 1-mediated fast and transient Ca(2+) release from intracellular stores. However, phospholipase C (PLC) gamma1 triggers a second (slower) wave of calcium release from intracellular stores, and it is this PLCgamma1-generated signal that is responsible for Ca(2+) entry. Surprisingly, FcepsilonRI (a high affinity receptor for IgE)-triggered mast cell degranulation depends on the first, sphingosine kinase-mediated Ca(2+) signal. These two pathways act independently because antisense knock down of either enzyme does not interfere with the activity of the other enzyme. Of interest, similar to PLCgamma1, SPHK1 translocates rapidly to the membrane after FcepsilonRI cross-linking. Here we also show that SPHK1 activity depends on phospholipase D1 and that FcepsilonRI-triggered mast cell degranulation depends primarily on the activation of both phospholipase D1 and SPHK1.

Human mast cells transmigrate through human umbilical vein endothelial monolayers and selectively produce IL-8 in response to stromal cell-derived factor-1 alpha.

Mature mast cells are generally considered to be less mobile cells residing within tissue sites. However, mast cell numbers are known to increase in the context of inflammation, and mast cells are recognized to be important in regulating local neutrophil infiltration. CXC chemokines may play a critical role in this process. In this study two human mast cell-like lines, HMC-1 and KU812, and human cord blood-derived primary cultured mast cells were employed to examine role of stromal cell-derived factor-1 (SDF-1) in regulating mast cell migration and mediator production. It was demonstrated that human mast cells constitutively express mRNA and protein for CXCR4. Stimulation of human mast cells with SDF-1, the only known ligand for CXCR4, induced a significant increase in intracellular calcium levels. In vitro, SDF-1 alpha mediated dose-dependent migration of human cord blood-derived mast cells and HMC-1 cells across HUVEC monolayers. Although SDF-1 alpha did not induce mast cell degranulation, it selectively stimulated production of the neutrophil chemoattractant IL-8 without affecting TNF-alpha, IL-1beta, IL-6, GM-CSF, IFN-gamma, or RANTES production, providing further evidence of the selective modulation of mast cell function by this chemokine. These findings provide a novel, SDF-1-dependent mechanism for mast cell transendothelial migration and functional regulation, which may have important implications for the local regulation of mast cells in disease.

TREATMENT OF OCULAR ALLERGY

Treatment of ocular hypersensitivity reactions requires a multidisciplinary approach in conjunction with an ophthalmologist and a clinical allergist/immunologist. Mast cell activation plays a central role in ocular allergy. Mast cells are found at a high density in normal conjunctiva (5000 per mm 3 ) 4 and their density increases in the more chronic forms of ocular hypersensitivity disorders. Treatment is directed at the release of preformed and newly formed mediators. Histamine, the prototype of the preformed mediators, has been shown to be elevated in tears of patients with vernal conjunctivitis. 1 Furthermore, these patients have an exaggerated response to histamine challenge compared with nonallergic controls. 15 This hyper-responsiveness of allergic patients to nonspecific stimuli also occurs in asthmatics. 22 Two types of histamine receptors have been delineated: 1 The H 1 receptor causes vasodilatation, increased vascular permeability, and itching. 2 H 2 receptors also cause vasodilatation and itching, and they alone are responsible for the mucous discharge seen in allergic eye disease. Newly synthesized mediators include derivatives of the eicosanoids, which are a class of potent oxygenated lipids that are generated from 20 carbon unsaturated fatty acids, usually arachidonic acid. Arachidonic acid is covalently bound (esterified) to membrane phospholipids. Upon stimulation of mast cells by allergen, arachidonic acid is hydrolyzed from membrane phospholipids and then oxygenated by cyclo-oxygenase (now known to be prostaglandin H synthase) to form prostaglandin H 2 (PGH 2 ). PGH 2 is then in turn the precursor of all prostaglandins, prostacyclins, and thromboxanes. The type of eiconsanoid ultimately synthesized depends on the enzymatic machinery present, and in mast cells, the major prostanoid produced is PGD 2 . Using quantitative gas chromatography and mass spectroscopy, Roberts et al have shown that isolated rat peritoneal and pleural mast cells produce PGD 2 and several other oxygenated products of arachidonic acid. Upon stimulation of these cells by ionophore, PGD 2 synthesis increases 10-fold, with modest increases in prostaglandins F 2 , E 2 , thromboxane B 2 , and a prostacyclin breakdown product (6-keto-prostaglandin F 1a ). 52 Stimulation of human mast cells by anti-IgE antibody also has been shown to lead to histamine release and PGD 2 biosynthesis. 44 PGD 2 has been shown to be elevated in tears of patients with vernal conjunctivitis, 25 and when instilled into human eyes leads to injection, chemosis, and eosinophil influx. 3